APTX acts in DNA double-strand break repair in a manner distinct from XRCC4.

Aprataxin (APTX), the product of the causative gene for hereditary neurogenerative syndromes Ataxia-oculomotor apraxia 1 and early onset ataxia with oculomotor apraxia and hypoalbuminemia, has an enzymatic activity of removing adenosine monophosphate from DNA 5'-end, which arises from abortive ligation by DNA ligases. It is also reported that APTX physically binds to XRCC1 and XRCC4, suggesting its involvement in DNA single-strand break repair (SSBR) and DNA double-strand break repair (DSBR) via non-homologous end joining pathway. Although the involvement of APTX in SSBR in association with XRCC1 has been established, the significance of APTX in DSBR and its interaction with XRCC4 have remained unclear. Here, we generated APTX knock-out (APTX-/-) cell from human osteosarcoma U2OS through CRISPR/Cas9-mediated genome editing system. APTX-/- cells exhibited increased sensitivity toward ionizing radiation (IR) and Camptothecin in association with retarded DSBR, as shown by increased number of retained γH2AX foci. However, the number of retained 53BP1 foci in APTX-/- cell was not discernibly different from wild-type cells, in stark contrast to XRCC4-depleted cells. The recruitment of GFP-tagged APTX (GFP-APTX) to the DNA damage sites was examined by laser micro-irradiation and live-cell imaging analysis using confocal microscope. The accumulation of GFP-APTX on the laser track was attenuated by siRNA-mediated depletion of XRCC1, but not XRCC4. Moreover, the deprivation of APTX and XRCC4 displayed additive inhibitory effects on DSBR after IR exposure and end joining of GFP reporter. These findings collectively suggest that APTX acts in DSBR in a manner distinct from XRCC4.

Implication of E3 ligase RAD18 in UV-induced mutagenesis in human induced pluripotent stem cells and neuronal progenitor cells.

Pluripotent stem cells (PSCs) have the potential to differentiate to any of the other organs. The genome DNA integrity of PSCs is maintained by a high level of transcription for a number of genes involved in DNA repair, cell cycle and apoptosis. However, it remains unclear how high the frequency of genetic mutation is and how these DNA repair factors function in PSCs. In this study, we employed Sup F assay for the measurement of mutation frequency after UV-C irradiation in induced pluripotent stem cells (iPSCs) as PSC models and neural progenitor cells (NPCs) were derived from iPSCs as differentiated cells. iPSCs and NPCs exhibited a lower mutation frequency compared with the original skin fibroblasts. In RNA-seq analysis, iPSCs and NPCs showed a high expression of RAD18, which is involved in trans-lesion synthesis (TLS) for the emergency tolerance system during the replication process of DNA. Although RAD18 is involved in both error free and error prone TLS in somatic cells, it still remains unknown the function of RAD18 in PSCs. In this study we depleted of the RAD18 by siRNA knockdown resulted in decreased frequency of mutation in iPSCs and NPCs. Our results will provide information on the genome maintenance machinery in PSCs.

THE ROLE OF DNA DOUBLE-STRAND BREAK REPAIR THROUGH NON-HOMOLOGOUS END JOINING IN THE DOSE-RATE EFFECT IN TERMS OF CLONOGENIC ABILITY.

It is generally and widely accepted that the biological effects of a given dose of ionizing radiation, especially those of low linear energy transfer radiations like X-ray and gamma ray, become smaller as the dose rate becomes lower. This phenomenon, known as 'dose-rate effect (DRE),' is considered due to the repair of sublethal damage during irradiation but the precise mechanisms for DRE have remained to be clarified. We recently showed that DRE in terms of clonogenic cell survival is diminished or even inversed in rodent cells lacking Ku, which is one of the essential factors in the repair of DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Here we review and discuss the involvement of NHEJ in DRE, which has potential implications in radiological protection and cancer therapeutics.

3D Organoid Culture Using Skin Keratinocytes Derived from Human Induced Pluripotent Stem Cells.

The keratinocytes are predominant cells in the epidermis of the human skin. To assess the cellular response of the keratinocytes to the genotoxic stress, we derived the skin keratinocytes from human induced pluripotent stem cells (iPSCs). Furthermore, three-dimensional (3D) organoid culture method is powerful tool to analyze the organ and tissue response against the genotoxic stress. Here we describe the method of 3D organoid culture using skin keratinocytes derived from human iPSCs.

α-glucosyl-rutin activates immediate early genes in human induced pluripotent stem cells.

Rutin is a natural flavonoid glycoside found in several vegetables and fruits such as buckwheat and onion. Rutin has a range of pharmacological effects that include anti-oxidant, anti-inflammation, anti-bacterial, and anti-cancer activities. α-glucosyl-rutin (AGR) is a derivative of rutin with increased water solubility that is used in cosmetics and foods. However, the effects of AGR on cellular responses have not been clarified, especially in stem cells. Induced pluripotent stem cells (iPSCs) show high proliferative activity and pluripotency; however, regulation of molecular machinery such as cell cycle, metabolism, and DNA repair differs between iPSCs and somatic cells. Here, we compared the effects of AGR on iPSCs and differentiated cells (fibroblasts and skin keratinocytes). AGR-treated iPSCs exhibited increased cell viability. RNA sequencing and reverse transcriptase PCR analysis revealed that AGR induced expression of immediate early genes (IEGs) and differentiation-related genes in iPSCs. Our results suggest that AGR may activate differentiation signals mediated by IEG responses in iPSCs, resulting in altered metabolic activity and increased cell viability.

DNA-Dependent Protein Kinase Catalytic Subunit: The Sensor for DNA Double-Strand Breaks Structurally and Functionally Related to Ataxia Telangiectasia Mutated.

The DNA-dependent protein kinase (DNA-PK) is composed of a DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Ku70/Ku80 heterodimer. DNA-PK is thought to act as the "sensor" for DNA double-stranded breaks (DSB), which are considered the most deleterious type of DNA damage. In particular, DNA-PKcs and Ku are shown to be essential for DSB repair through nonhomologous end joining (NHEJ). The phenotypes of animals and human individuals with defective DNA-PKcs or Ku functions indicate their essential roles in these developments, especially in neuronal and immune systems. DNA-PKcs are structurally related to Ataxia-telangiectasia mutated (ATM), which is also implicated in the cellular responses to DSBs. DNA-PKcs and ATM constitute the phosphatidylinositol 3-kinase-like kinases (PIKKs) family with several other molecules. Here, we review the accumulated knowledge on the functions of DNA-PKcs, mainly based on the phenotypes of DNA-PKcs-deficient cells in animals and human individuals, and also discuss its relationship with ATM in the maintenance of genomic stability.

Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity.

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4's DSB repair function in normal development.

Health effects triggered by tritium: how do we get public understanding based on scientifically supported evidence?

The Commission for 'Corresponding to Radiation Disaster of the Japanese Radiation Research Society' formulated a description of potential health effects triggered by tritium. This was in response to the issue of discharging water containing tritium filtered by the Advanced Liquid Processing System (ALPS), generated and stored in Fukushima Daiichi Nuclear Power Station after the accident. In this review article, the contents of the description, originally provided in Japanese, which gives clear and detailed explanation about potential health effects triggered by tritium based on reliable scientific evidence in an understandable way for the public, were summarized. Then, additional information about biochemical or environmental behavior of organically bound tritium (OBT) were summarized in order to help scientists who communicate with general public.

The FHA domain of PNKP is essential for its recruitment to DNA damage sites and maintenance of genome stability.

Polynucleotide kinase phosphatase (PNKP) has dual enzymatic activities as kinase and phosphatase for DNA ends, which are the prerequisite for the ligation, and thus is involved in base excision repair, single-strand break repair and non-homologous end joining for double-strand break (DSB) repair. In this study, we examined mechanisms for the recruitment of PNKP to DNA damage sites by laser micro-irradiation and live-cell imaging analysis using confocal microscope. We show that the forkhead-associated (FHA) domain of PNKP is essential for the recruitment of PNKP to DNA damage sites. Arg35 and Arg48 within the FHA domain are required for interactions with XRCC1 and XRCC4. PNKP R35A/R48A mutant failed to accumulate on the laser track and siRNA-mediated depletion of XRCC1 and/or XRCC4 reduced PNKP accumulation on the laser track, indicating that PNKP is recruited to DNA damage sites via the interactions between its FHA domain and XRCC1 or XRCC4. Furthermore, cells expressing PNKP R35A/R48A mutant exhibited increased sensitivity toward ionizing radiation in association with delayed SSB and DSB repair and genome instability, represented by micronuclei and chromosome bridges. Taken together, these findings revealed the importance of PNKP recruitment to DNA damage sites via its FHA domain for DNA repair and maintenance of genome stability.

Diminished or inversed dose-rate effect on clonogenic ability in Ku-deficient rodent cells.

The biological effects of ionizing radiation, especially those of sparsely ionizing radiations like X-ray and γ-ray, are generally reduced as the dose rate is reduced. This phenomenon is known as 'the dose-rate effect'. The dose-rate effect is considered to be due to the repair of DNA damage during irradiation but the precise mechanisms for the dose-rate effect remain to be clarified. Ku70, Ku86 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are thought to comprise the sensor for DNA double-strand break (DSB) repair through non-homologous end joining (NHEJ). In this study, we measured the clonogenic ability of Ku70-, Ku86- or DNA-PKcs-deficient rodent cells, in parallel with respective control cells, in response to high dose-rate (HDR) and low dose-rate (LDR) γ-ray radiation (~0.9 and ~1 mGy/min, respectively). Control cells and murine embryonic fibroblasts (MEF) from a severe combined immunodeficiency (scid) mouse, which is DNA-PKcs-deficient, showed higher cell survival after LDR irradiation than after HDR irradiation at the same dose. On the other hand, MEF from Ku70-/- mice exhibited lower clonogenic cell survival after LDR irradiation than after HDR irradiation. XR-V15B and xrs-5 cells, which are Ku86-deficient, exhibited mostly identical clonogenic cell survival after LDR and HDR irradiation. Thus, the dose-rate effect in terms of clonogenic cell survival is diminished or even inversed in Ku-deficient rodent cells. These observations indicate the involvement of Ku in the dose-rate effect.

Linker region is required for efficient nuclear localization of polynucleotide kinase phosphatase.

Polynucleotide kinase phosphatase (PNKP) is a DNA repair factor with dual enzymatic functions, i.e., phosphorylation of 5'-end and dephosphorylation of 3'-end, which are prerequisites for DNA ligation and, thus, is involved in multiple DNA repair pathways, i.e., base excision repair, single-strand break repair and double-strand break repair through non-homologous end joining. Mutations in PNKP gene causes inherited diseases, such as microcephaly and seizure (MCSZ) by neural developmental failure and ataxia with oculomotor apraxia 4 (AOA4) and Charcot-Marie-Tooth disease 2B2 (CMT2B2) by neurodegeneration. PNKP consists of the Forkhead-associated (FHA) domain, linker region, phosphatase domain and kinase domain. Although the functional importance of PNKP interaction with XRCC1 and XRCC4 through the FHA domain and that of phosphatase and kinase enzyme activities have been well established, little is known about the function of linker region. In this study, we identified a functional putative nuclear localization signal (NLS) of PNKP located in the linker region, and showed that lysine 138 (K138), arginine 139 (R139) and arginine 141 (R141) residues therein are critically important for nuclear localization. Furthermore, double mutant of K138A and R35A, the latter of which mutates arginine 35, central amino acid of FHA domain, showed additive effect on nuclear localization, indicating that the FHA domain as well as the NLS is important for PNKP nuclear localization. Thus, this study revealed two distinct mechanisms regulating nuclear localization and subnuclear distribution of PNKP. These findings would contribute to deeper understanding of a variety of DNA repair pathway, i.e., base excision repair, single-strand break repair and double-strand break repair.

Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells.

Pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a dual capability to self-renew and differentiate into all cell types necessary to develop an entire organism. Differentiation is associated with dynamic epigenetic alteration and transcriptional change, while self-renewal depends on maintaining the genome DNA accurately. Genome stability of PSCs is strictly regulated to maintain pluripotency. However, the DNA damage response (DDR) mechanism in PSCs is still unclear. There is accumulating evidence that genome stability and pluripotency are regulated by a transcriptional change in undifferentiated and differentiated states. iPSCs are ideal for analyzing transcriptional regulation during reprogramming and differentiation. This study aimed to elucidate the transcriptional alteration surrounding genome stability maintenance, including DNA repair, cell cycle checkpoints and apoptosis in fibroblasts, iPSCs and neural progenitor cells (NPCs) derived from iPSCs as differentiated cells. After ionizing radiation exposure, foci for the DNA double-stranded break marker γ-H2AX increased, peaking at 0.5 h in all cells (>90%), decreasing after 4 h in fibroblasts (32.3%) and NPCs (22.3%), but still remaining at 52.5% (NB1RGB C2 clone) and 54.7% (201B7 cells) in iPSCs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were detected, indicating that iPSCs' apoptosis increases. In addition, RNA sequencing (RNA-Seq) analysis showed high expression of apoptosis genes (TP53, CASP3 and BID) in iPSCs. Results suggested that increased apoptosis activity maintains accurate, undifferentiated genome DNA in the cell population.

DNA Damage Response After Ionizing Radiation Exposure in Skin Keratinocytes Derived from Human-Induced Pluripotent Stem Cells.

PURPOSE: Epidermal cells are positioned on the body surface and thus risk being exposed to genotoxic stress, including ionizing radiation (IR), ultraviolet rays, and chemical compounds. The biological effect of IR on the skin tissue is a significant problem for medical applications such as radiation therapy. Keratinocyte stem cells and progenitors are at risk for IR-dependent tumorigenesis during radiation therapy for cancer treatment. To elucidate the molecular mechanism of genome stability in epidermal cells, we derived skin keratinocytes from human-induced pluripotent stem cells (iPSCs) and analyzed their DNA damage response (DDR). METHODS AND MATERIALS: Skin keratinocytes were derived from iPSCs and designated as first- (P1), second- (P2), and third- (P3) passage cells to compare the differentiation states of DDR. After 2 Gy gamma-ray exposure, cells were immunostained with DNA double-strand break markers γ-H2AX/53BP1 and cell senescence markers p16/p21 for DDR analysis. DDR protein expression level, cell survival, and apoptosis were analyzed by western blotting, WST-8 assay and TUNEL assay, respectively. DDR of constructed 3D organoid modeling was also analyzed. RESULTS: P1, P2, and P3 keratinocytes were characterized with keratinocyte markers keratin 14 and p63 using immunofluorescence, and all cells were positive to both markers. Derived keratinocytes showed high expression of integrin α6 and CD71 (real-time (qRT)-PCR ratio: iPSCs: integrin α6: 1.12, CD71: 1.25, P1: integrin α6: 7.80, CD71: 0.43, P2: integrin α6: 5.53, CD71: 0.48), suggesting that P1 and P2 keratinocytes have potential as keratinocyte progenitors. Meanwhile, P3 keratinocytes showed low expression of integrin α6 and CD71 (qRT-PCR ratio: P3: integrin α6: 0.55, CD71: 0.10), suggesting differentiated keratinocytes. After IR exposure, the P1 and P2 keratinocytes showed an increase in DNA repair activity by a γ-H2AX/53BP1 focus assay (P1: γ-H2AX: 28.0%, 53BP1: 17.0%, P2: γ-H2AX: 37.7%, 53BP1: 28.3%) but not in P3 keratinocytes (P3: γ-H2AX: 74.7%, 53BP1: 63.7%) compared with iPSCs (γ-H2AX: 57.0%, 53BP1: 55.0%). Furthermore, in derived keratinocytes, expression of the cellular senescence markers p16 and p21 were increased compared with iPSCs (P16: non irradiated, iPSCs: 0%, P1: 12.5%, P2: 14.5%, P3: 29.7%, IR, iPSCs: 0%, P1: 19.5%, P2: 34.8%, P3: 64.5%). DDR protein expression, cellular sensitivity, and apoptosis activity decreased in derived keratinocytes compared with iPSCs. CONCLUSIONS: We have demonstrated the derivation of keratinocytes from iPSCs and their characterization of differentiated states and DDR. Derived keratinocytes showed progenitors like character as a result of DDR. These results suggest that derived keratinocytes are useful tools for analyzing the effects of IR, such as DDR on the skin tissue from radiation therapy for cancer.

Apurinic endonuclease-1 preserves neural genome integrity to maintain homeostasis and thermoregulation and prevent brain tumors.

Frequent oxidative modification of the neural genome is a by-product of the high oxygen consumption of the nervous system. Rapid correction of oxidative DNA lesions is essential, as genome stability is a paramount determinant of neural homeostasis. Apurinic/apyrimidinic endonuclease 1 (APE1; also known as "APEX1" or "REF1") is a key enzyme for the repair of oxidative DNA damage, although the specific role(s) for this enzyme in the development and maintenance of the nervous system is largely unknown. Here, using conditional inactivation of murine Ape1, we identify critical roles for this protein in the brain selectively after birth, coinciding with tissue oxygenation shifting from a placental supply to respiration. While mice lacking APE1 throughout neurogenesis were viable with little discernible phenotype at birth, rapid and pronounced brain-wide degenerative changes associated with DNA damage were observed immediately after birth leading to early death. Unexpectedly, Ape1Nes-cre mice appeared hypothermic with persistent shivering associated with the loss of thermoregulatory serotonergic neurons. We found that APE1 is critical for the selective regulation of Fos1-induced hippocampal immediate early gene expression. Finally, loss of APE1 in combination with p53 inactivation resulted in a profound susceptibility to brain tumors, including medulloblastoma and glioblastoma, implicating oxidative DNA lesions as an etiologic agent in these diseases. Our study reveals APE1 as a major suppressor of deleterious oxidative DNA damage and uncovers specific and broad pathogenic consequences of respiratory oxygenation in the postnatal nervous system.

Induction of Excess Centrosomes in Neural Progenitor Cells during the Development of Radiation-Induced Microcephaly.

The embryonic brain is one of the tissues most vulnerable to ionizing radiation. In this study, we showed that ionizing radiation induces apoptosis in the neural progenitors of the mouse cerebral cortex, and that the surviving progenitor cells subsequently develop a considerable amount of supernumerary centrosomes. When mouse embryos at Day 13.5 were exposed to γ-rays, brains sizes were reduced markedly in a dose-dependent manner, and these size reductions persisted until birth. Immunostaining with caspase-3 antibodies showed that apoptosis occurred in 35% and 40% of neural progenitor cells at 4 h after exposure to 1 and 2 Gy, respectively, and this was accompanied by a disruption of the apical layer in which mitotic spindles were positioned in unirradiated mice. At 24 h after 1 Gy irradiation, the apoptotic cells were completely eliminated and proliferation was restored to a level similar to that of unirradiated cells, but numerous spindles were localized outside the apical layer. Similarly, abnormal cytokinesis, which included multipolar division and centrosome clustering, was observed in 19% and 24% of the surviving neural progenitor cells at 48 h after irradiation with 1 and 2 Gy, respectively. Because these cytokinesis aberrations derived from excess centrosomes result in growth delay and mitotic catastrophe-mediated cell elimination, our findings suggest that, in addition to apoptosis at an early stage of radiation exposure, radiation-induced centrosome overduplication could contribute to the depletion of neural progenitors and thereby lead to microcephaly.

Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability.

Polynucleotide kinase-phosphatase (PNKP) is a DNA repair factor possessing both 5'-kinase and 3'-phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame-shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non-homologous end-joining, pathways required for repair of both DNA single- and double-strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease.

Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes.

DNA damage is considered to be a prime factor in several spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. We observed the endogenous accumulation of pathogenic topoisomerase-1 (Top1)-DNA cleavage complexes (Top1ccs) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We found that the defective DNA damage response factors in these two diseases cooperatively modulated Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single-strand break repair factors, including tyrosyl-DNA phosphodiesterase-1 or XRCC1, resulted in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Notably, direct Top1 poisoning to elevate Top1cc levels phenocopied the neuropathology of the mouse models described above. Our results identify a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating that genome integrity is important for preventing disease in the nervous system.

High LET radiation amplifies centrosome overduplication through a pathway of γ-tubulin monoubiquitination.

PURPOSE: Radiation induces centrosome overduplication, leading to mitotic catastrophe and tumorigenesis. Because mitotic catastrophe is one of the major tumor cell killing factors in high linear energy transfer (LET) radiation therapy and long-term survivors from such treatment have a potential risk of secondary tumors, we investigated LET dependence of radiation-induced centrosome overduplication and the underlying mechanism. METHODS AND MATERIALS: Carbon and iron ion beams (13-200 keV/µm) and γ-rays (0.5 keV/µm) were used as radiation sources. To count centrosomes after IR exposure, human U2OS and mouse NIH3T3 cells were immunostained with antibodies of γ-tubulin and centrin 2. Similarly, Nbs1-, Brca1-, Ku70-, and DNA-PKcs-deficient mouse cells and their counterpart wild-type cells were used for measurement of centrosome overduplication. RESULTS: The number of excess centrosome-containing cells at interphase and the resulting multipolar spindle at mitosis were amplified with increased LET, reaching a maximum level of 100 keV/µm, followed by sharp decrease in frequency. Interestingly, Ku70 and DNA-PKcs deficiencies marginally affected the induction of centrosome overduplication, whereas the cell killings were significantly enhanced. This was in contrast to observation that high LET radiation significantly enhanced frequencies of centrosome overduplication in Nbs1- and Brca1-deficient cells. Because NBS1/BRCA1 is implicated in monoubiquitination of γ-tubulin, we subsequently tested whether it is affected by high LET radiation. As a result, monoubiquitination of γ-tubulin was abolished in 48 to 72 hours after exposure to high LET radiation, although γ-ray exposure slightly decreased it 48 hours postirradiation and was restored to a normal level at 72 hours. CONCLUSIONS: High LET radiation significantly reduces NBS1/BRCA1-mediated monoubiquitination of γ-tubulin and amplifies centrosome overduplication with a peak at 100 keV/µm. In contrast, Ku70 and DNA-PKcs deficiencies mitigate centrosome overduplication, although deficiencies of both NBS1/BRCA1 and Ku70/DNA-PKcs markedly enhance cell killing.